7076
J. Org. Chem. 1995,60, 7076-7077
Communications Subglutinols A and B: Immunosuppressive Compounds from the Endophytic Fungus Fusarium subglutinans Julie C. Lee,t Emil Lobkovsky,? Nathan B. Pliam,* Gary Strobel,*,#and Jon Clardy*Bt Department of Chemistry, Cornell University, Ithaca, New York 14853-1301, Pharmagenesis, Inc., 3183 Porter Drive, Palo Alto, California 94304, and Department of Plant Pathology, Montana State University, Bozeman, Montana 5971 7
Table 1. NMR Data for Subglutinol A (1)in MeOD 13C a 1H b,c HMBCd ROESYe 1 26.2 t 1.56(qd, 13.0, 5.0), 1.45 2 2 32.1 t 2.45 (br td, 13.5,6.0), 1 , 4 , 19 2.05 (br dd, 14.0, 3.5)
3 149.9 s 4 5 6
56.5 d 39.6 s 35.9 t
7 8
23.3 t 88.6 d 45.5s 46.5d 49.0 t 75.9 d 128.4 d 136.4s 25.8 q 18.2 q 17.5 q 25.1 q
9 10
Received July 19, 1995 (Revised Manuscript Received September 11, 1995)
11 12 13
Immunosuppressive drugs are used today to prevent allografl rejection in transplant patients, and in the future they could be used to treat autoimmune diseases such as rheumatoid arthritis and insulin-dependent diabetes. Currently approved immunosuppressive agents such as cyclosporin A and FK506 possess some undesirable side effects, and the search for better immunosuppressive agents continues.2 We have been investigating endophytic fungi-the fungi that live in the intercellular spaces inside living plants-as a source of novel compounds. This search has emphasized fungi from plants with demonstrated biological activity, and in screening the fungi from the perennial twining vine Tripterygium wilfordii, we discovered two novel immunosuppressive compounds that form the basis of this report. T. wilfordii, which has been used in Chinese traditional medicine for over two thousand years, is familiar to chemists as the source of cytotoxic diterpene lactones such as tripdiolide3 and insecticidal alkaloids4 such as wilfordine. Recently, several group^^,^ have reported the isolation of immunosuppressive triterpenes from T.wilfordii. An endophytic fungus from T. wilfordii, identified as Fusarium subglutinans, produces the immunosuppressive but noncytotoxic diterpene pyrones subglutinol A (1)and B
14
15 16 17 18
2,4, 20 2, 18, 19 4, 18 7, 18
2.17 (dd, 11.5,4.5) 1.90 1.38 (dt, 14.0,3.5) 1.78, 1.74 3.17 (dd, 11.5, 4.0)
11,17 11,17 1.74 2,4,6, 17, 18 1.90, 1.45(dd, 11.5,4.0) 8, 17 4.75 (td, 9.5,3.5) 11 5.35 (br dt, 8.5, 1.5) 11, 15, 16 15,16 1.72 (d, 0.8) 13,16 1.69 (d, 1.0) 13,15 0.85 (s) 8, 11 0.96 (s) 4, 6, 10
19 110.7 t 22.7 t
4.50 (t, 2.5), 4.25 (t, 2.0) 2, 4 2.74 (dd, 13.5, 12.0), 2.58 (dd, 13.5,5.0) 21 103.7 s 20 22* 165.35s 20,27 23 109.0 s 26,27 24 156.8 s 26,27 25* 165.30s 20 26 17.3 q 2.19 ( 8 ) 27 10.4 q 1.91 (s)
0.96,4.25
0.85, 0.96 1.69,4.75
1.69,3.17 1.72,0.85 5.35 3.17 0.96,5.35 0.85,2.17, 4.25 0.96,2.17
20
a 13C spectrum was recorded at 100 MHz, referenced to d3MeOD at 49.0 ppm. Multiplicity was based on the DEFT spectrum. lH spectrum was recorded at 500 MHz, referenced to CHDzOD a t 3.30 ppm. Assignments to carbons were based on HMQC data. Those signals without indicated splitting patterns are either multiplets or buried within other signals. Numbers correspond to position of the proton. e The chemical shifts of the protons are given. Only those that are relevant to stereochemical assignment are shown.
(2).
F. subglutinans was cultured in modified M-1-D medium, and immunosuppressive activity, as judged by the mixed lymphocyte reaction, appeared in the ethyl acetate extract of the culture medium. The extract was chromatographed on silica gel using CH2C12-MeOH mixtures. The active fractions were subjected to reverse phase HPLC on a C-18 column with a solvent gradient of 75% MeOH-H20 to 100% MeOH to give two active compounds, subglutinol A (1)and B (2) (3:l). Compounds 1 (mp 185 "C dec; [a]% = -58.7" (c 0.027, MeOW) and 2 t
Department of Chemistry, Cornell University.
* Pharmagenesis, Inc.
Department of Plant Pathology, Montana State University. (1)Allison, A. C. In Immunosuppressive and Antiinflammatory Drugs; Allison, A. C., Lafferty, K. J., Fliri, H., Eds.; The New York Academy of Sciences: New York, 1993; Vol. 696; pp xi-xx. (2) Wiederrecht, G.; Lam, E.; Hung, S.; Martin, M.; Sigal, N. In Immunosuppressive and Antiinflammatory Drugs; Allison, A. C., Lafferty, K. J., Fliri, H., Eds.; The New York Academy of Sciences: New York, 1993; Vol. 696; pp 9-19. (3) Kupchan, S. M.; Court, W. A.; Dailey, R. G.; Gilmore, C. J.; Bryan, R. F. J . Am. Chem. SOC.1972,94, 7194-7195. (4) Shizuri, Y.; Yamada, K.; Hirata, Y. Tetrahedron Lett. 1973,741744. ( 5 ) Kutney, J. P.; Hewitt, G. M.; Lee, G.; Piotrowska, K.; Roberts, M.; Rettig, S. J. Can. J. Chem. 1992, 70, 1455-1469. (6) Li, X. Y. Int. J. Immunother. 1993, 9, 181-187.
1 Subglutinol A - 12 S
-
2 Subglutinol B 12 R
(mp 220 "C dec; [aIz5~ = -75.0" (c 0.288, MeOH) have identical W spectra, very similar 'H- and I3C-NMR spectra, and the same molecular formula, C27H3804, as judged by HRFABMS. The formula requires nine degrees of unsaturation, and since nine signals in the 13CNMR are sp2, the compounds contain four rings and a carbonyl (Table 1). A broad IR band at 3400-3100 cm-I and a strong absorption at 1664 cm-' indicate an alcohol, which is consistent with the exchangeable proton in the IH-NMR, and a conjugated carbonyl. The planar structure of the more abundant subglutinol A (1) can be deduced from analysis of one- and twodimensional NMR spectra (Table 1). Partial structures
0022-3263/95/1960-7076$09.00/00 1995 American Chemical Society
J . Org. Chem., Vol. 60, No. 22, 1995 7077
Communications
A
6
C
Figure 1. Fragments of 1 derived from COSY and HMBC
spectra.
A, B, and C for 1 (Figure 1)are obtained from analyses of the COSY, HMQC, and HMBC spectra as sketched below. In A, the position of the exocyclic double bond is determined from the heteronuclear correlations observed between the protons at 6 2.05 ((2-2)and 2.17 ((2-4) to the carbons at 6 149.9 ((2-3) and 110.7 (C-23). Because the protons of C-1 and C-2 are coupled, the methine signal at 6 46.5 (C-10)must have three-bond correlations to the protons a t 6 2.05 (C-2), 2.17 (C-4), 0.85 (C-171, and 0.96 ((3-18). The closure of ring A is established from the observed cross peaks of the signal at 6 39.6 (C-5)to the 6 2.17 (C-4)and 0.96 (C-18)resonances. In B,the olefinic signal at 6 5.35 ((2-13) shows a large coupling to 6 4.75 ((2-12) and long-range coupling to the terminal methyl groups. Since the quaternary carbon signal at 6 45.5 (C9) correlates to proton signals at 6 3.17 (C-8), 1.90 (C111,and 1.45 ((2-11,and the carbon signal at 6 35.9 (C-6) correlates to the proton signal a t 6 1.78 (C-7),A and B are connected to form the diterpene skeleton of 1. The remaining carbon resonances consist of five sp2, one methylene, and two methyl signals. Fragment C is partially constructed by signals at 6 109.0 and 156.8 having cross peaks to both methyl signals. Further analysis of the HMBC spectra indicate that the three remaining sp2 signals must be arranged to form a tautomeric a- or y-pyrone, since the methylene protons at 6 2.74 and 2.58 correlate with all three carbon resonances. Since A,, for 1 (293 nm) is significantly closer to the expected a-pyrone value,' and there is no literature precedent for a free 2-hydroxy-4-(y)-pyrone,1 is drawn as the a-pyrone tautomer. Finally, fragment B must contain a pentacyclic ether ring. Although no heteronuclear correlation is observed between the (2-12 and C-8 positions, a through-space correlation is observed in the ROESY spectrum of 1 (Figure 2). This NOE enhancement determines the relative stereochemistry a t C-8 and C-12 and forms the basis of the relative configurations at all of the other chiral centers as illustrated in Figure 2. NOE experiments indicate that subglutinol B (2) is the (2-12 epimer of subglutinol A (1) (Figure 2). A final check on the assigned stereochemistry is provided by a single crystal X-ray diffraction analysis of 2, which finally crystallized after the NMR experiments on 1 and 2 were completed.8 The results of this analysis are shown in Figure 3. In (7)Scott, A. I. Interpretation of the Ultraviolet Spectra of Natural Products; Pergamon Press: New York, 1964; p 140.
1
2
Figure 2. NOE correlations of 1 and 2.
Figure 3. A computer-generatedperspective drawing of the final X-ray model of subglutinol B (2). the solid state, compound 2 clearly exists as the a-pyrone,
not the y-pyrone, tautomer. The current analysis defines only the relative stereochemistry of compounds 1 and 2. The absolute configurations shown are drawn to agree with those established for the related compounds viridoxin A and B, antiinsecticidal metabolites of the entomopathogenicfungus Metarhizium fla~oviride.~ Compounds 1 and 2 are equipotent in the mixed lymphocyte reaction (MLR) assay and thymocyte proliferation (TP) assay (IC50 0.1 pM),and this equipotency suggests that the residue at C-12 does not interact with the biological target. In the same assay systems, cyclosporin A is roughly as potent in the MLR assay and lo4 more potent in the TP assay, but the lack of toxicity with 1 and 2 suggests that they definitely should be explored in greater detail. Further work on the in vivo activity of subglutinol A (1)and B (2) is in progress and will be reported in due course.
Acknowledgment. The research a t Cornel1 was supported by NIH CA2448'7 (J.C.), an NIH Training Grant Award (J.L.), and NCI grant lROl CA58315-03. Supporting Information Available: Spectral and physical data for subglutinol A (1)and B (2) (8 pages).
J0951304K (8)Space group P212121 with a = 7.187(1), b = 9.685(2), and c = 34.405(7)A and one molecule in the asymmetric unit. The final R-factor is 0.075 for the 2a data set. (9) Gupta, S.; Krasnoff, S. B.; Renwick, J. A. A.; Roberts, D. W.; RiosSteiner, J.; Clardy, J. J. Org. Chem. 1993,58, 1062-1067.
